In researching target protein interactions using an affinity resin, it is important to determine whether an affinity-bound protein is specific for a ligand or non-specific. Traditionally, to accomplish this purpose, an antagonism experiment that comprises adding a non-modified subject ligand to a starting material protein mixture in advance or simultaneously with the addition of the affinity resin, and confirming the reduction or disappearance of the amount of the subject protein, has been used. Hence, inhibition of the binding of the protein to the affinity resin by the co-presence of the ligand as an antagonist has been considered to be an essential condition for the determination that the protein is specific for the ligand. However, when applying this method, it is often difficult to dissolve a required amount of ligand in the subject protein mixture. This represents representing a drawback in that such experiments are substantially unperformable. In particular, when a pharmaceutical is the subject, which often possesses fat solubility (in particular, orally administrable pharmaceuticals possess fat solubility to ensure membrane permeability by passive diffusion), a sufficient ligand concentration cannot be achieved so that experimental studies of proteins found on affinity resins by antagonism experiments have been abandoned to date. Specifically, to perform an antagonism experiment, it is necessary to dissolve several hundred μg/ml of a ligand (for example, provided that TOYO-Pearl 10 μl=1 μmol is used, in the case of a ligand (antagonist) having a molecular weight of 500, a solubility of not less than 0.5 mg/ml is required, even when an equal amount of drug is present with the ligand on the resin) in an aqueous solution wherein the protein is present; generally, it is difficult to dissolve such a high concentration of ligand in a biological material solution wherein considerable amounts of various ions and solutes such as proteins are dissolved. This limitation is a problem common not only to pharmaceuticals but also to compounds that exhibit interesting pharmacological action in oral administration, for example, environmental substances, toxic substances and the like, and there has been a demand to overcome this limitation in the entire research into drug discovery target search.
Also, in conventional methods, a ligand is often added in an amount not less than the amount of the ligand on the resin to secure an antagonistic effect, and this practice is a major problem of protein denaturation due to the presence of a ligand at a high concentration of several mg/ml in a biological material solution such as a lysate. That is, even if band disappearance due to the addition of a ligand is observed during an antagonism experiment performed to determine the specificity of an affinity-resin-bound protein, it is difficult to determine whether the observation is due to antagonistic effect or derived from the inactivation of the protein by the non-specific protein-denaturing effect of the ligand.
Therefore, there has been a method of determining the ligand specificity of a protein that binds to an affinity resin, which enables solving 1) the problem of the solubility of subject ligand, and 2) the problem of the non-specific protein-denaturing effect of the subject ligand added, which have been problematic in conventional antagonism experiments described above.
It is an object of the present invention to provide a method of determining the ligand specificity of a protein that binds to an affinity resin, particularly to provide a method of determining ligand specificity wherein the solubility of ligand and the non-specific protein-denaturing effect of the added subject ligand are at issue.